Cysteine proteases such as calpain use a cysteine residue in their catalytic mechanism in contrast to serine proteases which utilize a serine residue. Cysteine proteases include papain, cathepsin B, calpains, and several viral and parasite enzymes. Neural tissues, including brain, are known to possess a large variety of proteases, including at least two calcium stimulated proteases termed calpains. Calpains are ubiquitous cytosolic proteolytic enzymes involved in both physiological and pathological cellular functions. Limited activation of calpains results in modification or activation of protein receptors, enzymes, and cytoskeletal proteins. Pathological cellular insults lead to more generalized calpain activation, resulting in cytoskeletal degradation and cell death. Calpain activation likely occurs due to sustained elevation of intracellular calcium that is a common feature of models of neuronal injury (Bartus, “The calpain hypothesis of neurodegeneration: evidence for a common cytotoxic pathway,” Neuroscientist 3:314-27, 1997, which is incorporated by reference herein in its entirety).
While calpains degrade a wide variety of protein substrates, cytoskeletal proteins seem to be particularly susceptible to attack. In some cases, the products of the proteolytic digestion of these proteins by calpain are distinctive and persistent over time. Since cytoskeletal proteins are major components of certain types of cells, this provides a simple method of detecting calpain activity in cells and tissues. Thus, calpain activation can be measured indirectly by assaying the proteolysis of the cytoskeletal protein spectrin, which produces a large, distinctive and biologically persistent breakdown product when attacked by calpain (Siman et al., Proc. Natl. Acad. Sci. USA 81:3572-76, 1984, which is incorporated by reference herein in its entirety). Activation of calpains and/or accumulation of breakdown products of cytoskeletal elements have been observed in neural tissues of mammals exposed to a wide variety of neurodegenerative diseases and conditions. For example, these phenomena have been observed following ischemia in gerbils and rats, following stroke in humans, following administration of the toxins kainate, trimethyltin or colchicine in rats, and in human Alzheimer's disease. In the nervous system, calpain activation is believed to be responsible for the calcium-mediated cell injury seen in ischemic stroke, spinal cord injury, closed head injury, Wallerian degeneration, ALS, and peripheral neuropathy.
Calpains are present, in many tissues in addition to the brain. Calpain I is activated by micromolar concentrations of calcium while calpain II is activated by millimolar concentrations. In the brain, calpain II is the predominant form, but calpain I is found at synaptic endings and is thought to be the form involved in long term potentiation, synaptic plasticity, and cell death. Other Ca2+ activated cysteine proteases may exist, and the term “calpain” is used to refer to all Ca2+ activated cysteine proteases, including calpain I and calpain II. The terms “calpain I” and “calpain II” are used herein to refer to the micromolar and millimolar activated calpains, respectively, as described above.
Cathepsin B, cathepsin L, cathepsin S, and other cathepsins are involved in muscular dystrophy, myocardial tissue damage, tumor metastasis, and bone resorption. In addition, a number of viral processing enzymes, which are essential for viral infection, are cysteine proteases. Inhibitors of cysteine proteases would thus have multiple therapeutic uses.
Numerous inhibitors of calpain and other cysteine protease have been described in the literature. These include peptide aldehydes such as Ac-Leu-Leu-Nle-H and leupeptin (Ac-Leu-Leu-Arg-H), as well as epoxysuccinates such as E-64. These compounds are not especially useful at inhibiting calpain in neural tissue in vivo because they are poorly membrane permeant and, accordingly, are not likely to cross the blood brain barrier very well. Also, many of these inhibitors have poor specificity and will inhibit a wide variety of proteases in addition to calpain. In addition, other classes of compounds that inhibit cysteine proteases include peptide diazomethyl ketones (Rich, In Protease Inhibitors, Barrett and Salvesen, Eds., Elsevier, N.Y., 1986, pp 153-78, which is incorporated by reference herein).
Potent calpain inhibitors are in general either transition-state analogs or irreversible covalent inhibitors. The active site of a cysteine protease contains Cys and His residue along with the so-called oxyanion hole. The mechanism of substrate hydrolysis involves the attack of the active site Cys on the scissile peptide bond. The active site cysteine of the enzyme adds to the scissile amide carbonyl group to form a tetrahedral adduct forming an oxyanion that interacts with the so called oxyanion hole. Subsequently, the tetrahedral adduct forms an acyl enzyme intermediate, which is then cleaved to the two hydrolysis products. Transition-state analog inhibitors such as peptide aldehydes, ketones, and α-ketoamides reversibly inhibit cysteine proteases by forming a hemithioacetal through reaction with the active site cysteine thiol group. This resembles the tetrahedral transition state involved in normal peptide substrate hydrolysis. Transition state inhibitors are very potent reversible inhibitors for cysteine proteases, since the enzyme has evolved to bind the tetrahedral transition state effectively.
Additional references to calpain inhibitors in the literatures are Tsujinaka et al., “Synthesis of a new cell-penetrating calpain inhibitor (calpeptin),” Biochem. Biophys. Res. Commun. 153:1201-08, 1988; Huang et al., “Amide derivatives of E64c as inhibitors of platelet calpains,” J. Med. Chem. 35:2048-54, 1992; Powers et al., “Irreversible inhibitors of serine, cysteine, and threonine proteases,” Chem. Rev. 102:4639-750, 2002; Powers, “Calpain Inhibitors,” In Design of Enzyme Inhibitors as Drugs,” Sandler and Smith, Eds., Oxford University Press, Oxford, 1994; Vol. 2, pp 754-66; Donkor, “A survey of calpain inhibitors,” Curr. Med. Chem. 7:1171-88, 2000; Krauser and Powers, “Calpain,” In Proteinase and Peptidase Inhibition: Recent Potential Targets for Drug Development, Smith and Simons, Eds., Taylor & Francis, New York, 2000, pp. 127-53, each of which are incorporated by reference herein in their entireties.
Dipeptidyl and tripeptidyl α-ketoesters, α-ketoamides and α-ketoacid transition-state inhibitors of calpains I and II have been reported in the literature. See for example Li et al., “Novel peptidyl α-ketoamide inhibitors of calpains and other cysteine proteases,” J. Med. Chem. 39:4089-98, 1996; Li et al., “Peptide α-ketoester, α-ketoamide and α-ketoacid inhibitors of calpains and other cysteine proteases,” J. Med. Chem. 36:3472-80, 1993, each of which are incorporated herein by reference in their entities. The composition in these publications are also disclosed in several patents: U.S. Pat. No. 5,514,694, issued May 7, 1996, to Powers et al.; U.S. Pat. No. 5,610,297, issued Mar. 11, 1997, to Powers; U.S. Pat. No. 5,650,508, issued Jul. 22, 1997, to Powers; U.S. Pat. No. 5,763,576, issued Jun. 9, 1998, to Powers; and U.S. Pat. No. 6,235,929, issued May 22, 2001, to Powers, each of which are incorporated herein by reference in their entireties.
One of the ketoamides, AK295, is a potent transition-state inhibitor for calpain I and II, but does have some inhibitory activity toward other cysteine proteases such as cathepsins (Li et al., J. Med. Chem. 36:3472-80, 1993). The ketone carbonyl group of AK295 is the group which forms the hemithioacetal with the active site cysteine of calpain. Since the enzyme has evolved to bind the tetrahedral transition state effectively, transition state inhibitors are usually potent reversible inhibitors for cysteine proteases. Reversibility occurs when the hemiacetal breaks down to reform the carbonyl compound and free enzyme.
No effective therapy has yet been developed for most neurodegenerative diseases and conditions, and other disease related to uncontrolled cysteine protease action. Millions of individuals suffer from neurodegenerative diseases, and thus there is a need for therapies effective in treating and preventing these diseases and conditions. There is a need for new compositions to treat other diseases related to cysteine protease activity. Further, because neuronal pathologies have a dramatic impact on quality of life of patients, there is a need for compositions and methods for treating these disorders, in particular, compositions and methods for treating pathologies with little or reduced side effects such as neuropathy. Thus, there is also a need for compositions and methods of treating other pathologies related to calpain activation. There is still another need for methods and compositions for disease resulting from other cysteine proteases particularly parasitic diseases. The compositions and methods disclosed herein address these and other needs.